PROJECT SUMMARY Traumatic brain injury (TBI) remains a significant cause of morbidity and mortality in the U.S., with a continued call for more effective interventions to improve outcome. Many compounds have shown promise in pre-clinical studies, but fail to translate effectively into the clinic. Our long-term goal is to improve TBI therapeutic translation through temporal and pathobiological specificity in administering compounds to individual patients. Here we postulate that certain therapeutics are effective only when appropriately timed to a specific post-TBI biochemical event. This application will specifically focus on TBI-induced dysfunction of potassium/chloride cotransporter 2 (KCC2), a neuron specific transporter that maintains the low intracellular chloride concentration essential to gabaergic inhibitory transmission. Aim 1a interrogates the post-translational modification of KCC2 ahead of its destabilization and triggered loss of the active dimeric form and associated binding partners. KCC2 functional cycling is post-translationally regulated and our preliminary data point to early modification preceding its delocalization. Aim 1b assesses the therapeutic potential of CLP290, a recently reported selective modulator of KCC2 that enhances preservation of its functional state at the plasma membrane, as observed in our preliminary studies following TBI. Effective administration of CLP290 will be tested in relation to post-translational triggers of KCC2 destabilization to underscore the mechanistic relevance of our preliminary data demonstrating therapeutic efficacy when given one day, but not immediately or two days following injury. Aim 2a will test the correlation of improved behavioral recovery with CLP290 administration and the functional preservation of chloride homeostasis, as critical to maintaining inhibitory neurotransmission. Aim 2b will evaluate whether CLP290 further reduces secondary tissue loss through maintenance of chloride homeostasis and associated inhibitory transmission counteracting excitotoxicity. With the successful completion of these studies, we expect to establish the mechanistic framework for the novel therapeutic targeting of KCC2 dysfunction following TBI. Furthermore, these studies will demonstrate the need to temporally correlate therapy with post-translational proteomic events that may then be targeted for selective biomarker development. We expect results from these studies will progress the theragnostics approach to TBI interventions. Specifically, these studies aim to treat the disruption of the excitatory/inhibitory imbalance in surviving tissues for improved functional recovery and potential alleviation of post-traumatic seizure. As well, the successful completion of the proposed training plan and project will provide Mr. Lizhnyak with exceptional preparation in pursuing an independent, productive research career.